Kinetics of methylation and binding of DNA by the EcoRV adenine-N6 methyltransferase.
The EcoRV DNA methyltransferase (M.EcoRV) specifically methylates the first adenine within its recognition sequence GATATC. Methylation rates of DNA by this enzyme are strongly influenced by the length of oligonucleotide substrates employed. If substrates >20 bp compared to a 12mer substrate, the kcat/Km increases 100-fold, although the enzyme does not contact more than 12 base-pairs on the DNA. Single-turnover rates are higher than kcat values. M.EcoRV binding to DNA is fast but dissociation from the DNA is slow, demonstrating that the multiple-turnover rate is limited by the rate of product release. The kinetics of DNA binding by M.EcoRV are not in accordance with the thermodynamics binding constant, suggesting that the M.EcoRV-DNA complex is involved in a slow conformational change. The salt dependence of DNA binding is different for non-specific substrates (d ln(KAss)/d ln(cNaCl) = - 2, indicative of electrostatic interactions) and specific substrates (d ln(KAss)/d ln(cNaCl) = + 1, indicative of hydrophobic interactions). This result demonstrates that the M.EcoRV-DNA complex has a different conformation in both binding modes. M.EcoRV does not discriminate between hemimethylated and unmethylated substrates. Using the 20mer we have analyzed the temperature and pH dependence of the single-turnover rate constant of M.EcoRV-DNA methylation by M.EcoRV has an activation energy of 40 kJ/mol and its rate increases with increasing pH. The pH dependence reveals the presence of an ionizable residue with a pKa of 7.9, which must be unprotonated for catalysis. The rates of DNA methylation remain unchanged if an abasic site is introduced instead of the thymidine residue that is base-paired to the target adenine, demonstrating that flipping out the target adenine cannot contribute to the rate-limiting step of the enzymatic reaction.
Jeltsch
A
A
Institut für Biochemie, Fachbereich Biologie, Justus-Liebig Universität, Heinrich-Buff Ring 58, Giessen, 35392, Germany.
Friedrich
T
T
Roth
M
M
D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019175:DNA Methylation; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009841:Oligonucleotides / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity; D013696:Temperature
D009841:Oligonucleotides; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific